A Yeast Phosphofructokinase Insensitive to the Allosteric Activator Fructose 2,6-Bisphosphate

Abstract

In this work we used in vitro mutagenesis to modify the allosteric properties of the heterooctameric yeast phosphofructokinase. Specifically, we identified two amino acids involved in the binding of the most potent allosteric activator fructose 2,6-bisphosphate. Thus, Ser 724 was replaced by an aspartate and His 859 by a serine in each of the enzyme subunits. Whereas the substitutions had no drastic effects when introduced only in one of the two types of subunits, kinetic parameters were modified when both subunits carried the mutation. Thus, the enzyme with His 859 3 Ser showed an increase in K a for binding of the activator, whereas the one with Ser 724 3 Asp failed to react to the addition of fructose 2,6-bisphosphate, at all. The enzymes still responded to other allosteric activators, such as AMP. Stabilities of the mutant subunits were not significantly altered in vivo, as judged from Western blot analysis. Phenotypically, strains expressing the mutant PFK genes showed a pronounced effect on the level of intermediary metabolites after growth on glucose. Mutants not responding to the activator at all (Ser 724 3 Asp) also displayed higher generation times on glucose medium. This could be suppressed by increasing the gene dosage of the mutant alleles. These results indicate that fructose 2,6-bisphosphate through its activation of phosphofructokinase plays an important role in regulation of the glycolytic flux.

Single Point Mutations in Either Gene Encoding the Subunits of the Heterooctameric Yeast Phosphofructokinase Abolish Allosteric Inhibition by ATP

“…Taken together, this strongly suggests that ATP can be bound at the catalytic site in the N-terminal half as well as at an allosteric site in the Cterminal half of each yeast Pfk subunit. Interestingly, Pro-728 is located only four amino acids downstream of Ser-724 in the deduced protein sequences of each subunit, which was shown to be involved in fructose 2,6-bisphosphate binding (21). These findings confirm a hypothesis by Poorman et al (20), who proposed that eukaryotic Pfk subunits evolved by a gene duplication event and that domains formerly serving catalytic functions evolved in the C-terminal duplicate to allosteric binding sites.…”

“…A hypothesis originally brought up by Poorman et al (20) attributes the origin of eukaryotic Pfk subunits to a gene duplication event, with the N-terminal half of each subunit retaining catalytic function and the C-terminal half evolving allosteric binding sites. This assumption was substantiated by our findings, when we abolished activation of yeast Pfk by fructose 2,6-bisphosphate using site-directed in vitro mutagenesis in sequences encoding the C-terminal halves of each subunit (21).…”

“…After resuspension in 1.5 ml of distilled water, cell debris was separated by a 15-min centrifugation in a microcentrifuge and the supernatant was used for NADP-or NADH-coupled enzymatic determinations. Fructose 2,6-bisphosphate levels were determined via activation of pyrophosphate-dependent phosphofructokinase from potatoes as described previously (21). Calculations were based on the assumption that an A 600 of 1 equals 0.4 mg/ml cell dry weight (34).…”

“…Sequencing of this region showed a single base substitution at position 2184, leading to an exchange of proline 728 for a leucine in the deduced amino acid sequence. It should be noted that this exchange is close to a serine residue (position 724) previously identified as being involved in the binding of the allosteric activator fructose 2,6-bisphosphate (21).…”

“…They were placed in a water bath at 50°C, and aliquots were taken at the time points indicated. Activities measured in 50 mM potassium phosphate buffer, pH 7.0, with 10 mM MgCl 2 at 1 mM ATP, 2.0 mM fructose 6-phosphate, and 10 M fructose 2,6-bisphosphate were set at 100% at time 0. the subunits (21). Here, we localized a mutation obtained in the PFK1-encoded ␣-subunit by classical genetic screening (22) as abolishing ATP inhibition also in the C-terminal half of the subunit (P728L).…”